Vertical zonation and heat tolerance of three littorinid gastropods on a rocky shore at Tanjung Chek Jawa, Singapore

2009·0.1 MB·English

by  Lee, Su-Li

THE RAFFLES BULLETIN OF ZOOLOGY 2009
THE RAFFLES BULLETIN OF ZOOLOGY 2009 57(2): 551–560
Date of Publication: 31 Aug.2009
© National University of Singapore
VERTICAL ZONATION AND HEAT TOLERANCE OF THREE LITTORINID
GASTROPODS ON A ROCKY SHORE AT TANJUNG CHEK JAWA, SINGAPORE
Su-Li Lee
Anglo-Chinese Junior College, 25 Dover Close East, Singapore 139745, Republic of Singapore
Shirley S. L. Lim
Ecology Laboratory, Natural Sciences & Science Education, National Institute of Education,
Nanyang Technological University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore
Email: [email protected] (Corresponding author)
ABSTRACT. – Three common rocky shore littorinid taxa, i.e., Littoraria spp. (a collective term for L.
strigata and L. articulata), Echinolittorina malaccana and E. vidua occupied different tidal heights based
on fi eld observations carried out at Tanjung [=Cape] Chek Jawa, Pulau [=Island] Ubin, Singapore in 2002.
Littoraria spp. were consistently observed at a lower level on the shore than E. malaccana and E. vidua.
Manually translocated littorinids returned to their preferred zones in the fi eld, i.e., E. malaccana returned
to the region above the MHWS level of 2.7 m while Littoraria spp. remained below the region occupied
by E. malaccana. Further, E. malaccana individuals with their shell nodules removed by fi ling did not
occupy a lower zone than intact conspecifi cs in the fi eld. Although signifi cant temperature differences (TD)
were observed between rock and shell surfaces in the laboratory (TD ≈ TD < TD ),
Littoraria fi led E. malaccana E. malaccana
preference for a particular height on the shore is probably governed more by physiological factors (e.g.,
heat tolerance) than by morphological adaptation (e.g., shell morphology). As the upper shore is exposed to
the sun for longer periods of time, it is expected that the heat tolerance of inhabitants of the higher shore
would be greater than those occupying the lower regions. Results of a 1-hour lethal temperature experiment
support this hypothesis: the temperature at which 50% mortality (1h LT ) was observed was highest for
50
E. malaccana (50.4°C) compared to E. vidua, (48.1 °C) and Littoraria spp. (47.5°C), possibly refl ecting
their relative tidal positions on the shore. Enzyme stability may also account for high heat tolerance of E.
malaccana. Incubation of the enzyme, glutamate oxaloacetate transaminase (GOT) at 55°C showed that E.
malaccana sustained high GOT activity compared to those of Littoraria spp. and E. vidua which decreased
sharply at the same temperature, again suggesting inherent physiological adaptation in E. malaccana.
KEY WORDS. – Zonation; heat tolerance; enzyme stability; Littoraria sp.; Echinolittorina malaccana;
Echinolittorina vidua.
INTRODUCTION spanning many days (McMahon, 1990). Desiccation
may be overcome by behavioural adaptations such as the
The intertidal rocky shore is universally divided into withdrawal of the foot and closure of the shell opening
zones according to characteristic intertidal fl ora and fauna with an operculum to reduce evaporation. Attachment to
(Raffaelli & Hawkins, 1996): the supralittoral, midlittoral the substratum using a mucous sheet – the mucous holdfast
and sublittoral zones. Such zonation is due to the stresses of (Bingham, 1972; Lim, 2008), further ensures that they will
varying intensities along the height of the shore. Higher levels not be dislodged easily or maintain minimal contact with the
of the shore (e.g., supralittoral zone) are exposed to the air and heated substratum when out of water. Survival in prolonged
sun for a longer period of time when compared with lower aerial exposure also depends on physiological adaptations
shore levels (Newell, 1979; Cleland & McMahon, 1989). such as the reduction of metabolic rates, e.g., in Littorina
Despite the extreme physical conditions in the supralittoral saxatilis (see McMahon & Russell-Hunter, 1977) and
zone, many gastropod species have adapted successfully to elevated thermal tolerance, e.g., in Nodilittorina pyramidalis,
life in this harsh zone. Gastropods living above the high tide N. granularis and Littorina brevicula (see Fraenkel, 1966)
mark are especially vulnerable to desiccation and thermal as well as Austrolittorina unifasciata (= N. unifasciata; see
stress, as they typically experience prolonged aerial exposure Reid, 2007) (see McMahon, 1990).
551
Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids
Common littorinids found on the rocky shores of Singapore in the cell (Cooper & Meister, 1985). The metabolic
include Littoraria articulata (Philippi, 1846), L. strigata importance of GOT is that it brings about a free exchange
(Philippi, 1846) (often as Littorina undulata in previous of amino groups between glutamate and aspartate; both of
literature) and Echinolittorina malaccana (Philippi, 1847) which are required for separate but essential steps in the
(also referred to in the literature variously as Nodilittorina urea cycle for ammonia detoxication and nitrogen excretion.
pyramidalis or N. trochoides; see Reid, 2007). A less The free movement of nitrogen between the glutamate and
common rough periwinkle, Echinolittorina vidua (Gould, aspartate pools is an important balancing process that is vital
1859) (previously Nodilittorina vidua; see Reid, 2007) is for normal cell metabolism (Cooper & Meister, 1985).
also found in certain rocky areas in Singapore.
Two previous littorinid distribution studies have been MATERIAL AND METHODS
conducted in Singapore on the south-eastern coast of
Singapore; one at a man-made breakwater (see Lam, 1980) Distribution studies
and the other at a monsoon drain (see Tan, 1988). To date,
there is no published literature with quantitative data on Two distribution studies were carried out at the rocky
the vertical distribution of Littoraria spp., Echinolittorina shore of Tanjung Chek Jawa on Pulau Ubin (approx. 1°
malaccana and E. vidua at natural rocky shores in Singapore. 24’N, 103° 59’E), an island off the north-eastern coast of
The purpose of the fi rst part of this study was to determine Singapore. The mean high water spring tide level (MHWS)
the zonation pattern of these littorinids at the rocky shores at the study site is 2.7 m (Hydrographic Department, 2001).
of Tanjung [=Cape] Chek Jawa, Pulau [=Island] Ubin, These studies were made at (i) a vertical rock and (ii) a
Singapore, through a distribution survey. sloping rock (angle of inclination ≈ 35.6°). The vertical
rock is located further inland (i.e., more up-shore in a little
A translocation experiment involving Littoraria spp. and ‘cove-like’ recess) as compared to the sloping rock located
E. malaccana was also carried out to verify the zonation at a small spit jutting out seawards. Hence, the vertical rock
pattern observed. Dislodged littorinids are able to crawl surface studied is exposed earlier by the receding tide and
upwards to their original zone, e.g., N. exigua (see Ohgaki, inundated later by the incoming tide when compared to the
1988a), Littorina littorea (see Petraitis, 1982) and Littorina sloping rock. Both rocks have sparse algal cover and a fair
unifasciata (see Chapman, 1999). It is hypothesized that number of crevices.
displaced Littoraria spp. and E. malaccana individuals in
this study would return to their preferred zones. Reid (1986) reported that the two closely related species
Littoraria articulata and L. strigata possess shells with
Shell sculpturing was reported by Vermeij (1973) to reduce similar shape, colour and banding pattern, and without
the relative effective areas for absorption, i.e., area in a plane examination of internal anatomical features, it was diffi cult
that is perpendicular to incident sun’s rays. This implies that to ascertain their identities in the fi eld. Thus, these two
there is greater heat loss by refl ection of solar radiation by littorinid species are collectively referred to as Littoraria
an uneven surface, thereby reducing heat absorption. The spp. in this study as dissection of every individual
shell of E. malaccana is nodulated while that of Littoraria encountered was neither feasible nor possible. Data from the
spp. individuals is smooth. Since E. malaccana occupies vertical rock were collected during three sampling sessions:
a higher region of the shore, the nodulation may be an June/July, September and December 2002, with a total
advantage for survival under prolonged aerial exposure. of 10 transect belts laid at each session. The distribution
The increased surface area of the shell due to the presence surveys were all carried out during three to fi ve days of
of nodules could facilitate convection and heat loss from consecutive spring tides. Accessibility to the study site
the shell. To test this hypothesis, the nodulated shells of was only possible when the tides were suitably low. Each
E. malaccana individuals were fi led down and included belt transect consisted of a transparent 10 cm-wide plastic
in the translocation study to elucidate the possible role of strip with a measuring tape attached. The position of each
nodulation in zonation. The effect of nodulation on heat Littoraria spp. (collectively Littoraria) and Echinolittorina
refl ection was tested both in the fi eld (for E. malaccana and malaccana (E. malaccana) individual encountered beneath
Littoraria spp.) and in the laboratory (for E. malaccana, the strip was recorded to the nearest 0.5 cm. Distribution
both intact and fi led, and Littoraria spp.). The temperature of Littoraria, E. malaccana and E. vidua was similarly
difference between rock and shell surfaces was used as the determined at the sloping rock on 28 and 29 August 2002.
response variable. A region covered by a total of 18 belt transects, each 15
cm in width, was surveyed. The tide level (TL) at the top
Two laboratory experiments were conducted to establish of the vertical rock is 0.1 m above MHWS.
possible physiology-related reasons for the zonation
pattern: (1) a heat tolerance test, represented by 1 hour
lethal temperatures (1h LT ), of the littorinids, and (2) a Translocation experiment
50
heat stability test of the enzyme, glutamate oxaloacetate
transaminase (GOT), extracted from these littorinids. The A translocation experiment was conducted in December
enzyme GOT, also known as aspartate aminotransferase, 2002. Thirty individuals of Littoraria and 60 E. malaccana
was chosen because it is one of the most active enzymes individuals were collected and transported to the laboratory
552
THE RAFFLES BULLETIN OF ZOOLOGY 2009
in a container (with seawater from the study site) for colour- would not be confounded by other factors, e.g., shade or
coding with nail polish. The nodules of 30 E. malaccana refl ection of heat from adjacent surfaces.
adult individuals were fi led off (henceforth termed “fi led
E. malaccana”) using an ordinary machinist’s file. All The temperature difference (TD) between rock surface and
experimental snails were returned to the study site within that of the shell surface (i.e., TD = T – T )
rock surface shell surface
two days to minimize stress and ensure their survival during for E. malaccana and Littoraria were tested separately for
the translocation experiment. Based on the distribution signifi cance using t-tests. TD and TD were
Littoraria E. malaccana
patterns observed from previous sampling periods, the then compared using the Mann-Whitney test statistic, as
MHWS level (2.7 m) was found to approximately separate there was non-normality in the data set. In the laboratory,
the regions occupied by Littoraria and E. malaccana. 25 E. malaccana, 25 fi led E. malaccana and 25 Littoraria
Thus, the marked littorinids were returned to the 2.7 m were used in an experiment involving heat treatment. Five
TL with the MHWS level clearly marked at the study E. malaccana, fi ve fi led E. malaccana and fi ve Littoraria
site. Crowe & Underwood (1999) suggested that a snail’s were placed on a piece of relatively smooth rock that
behaviour changed by merely dislodging it from its original most resembled that at the fi eld site. The rock was then
position. This may affect the results of the translocation placed approximately 20 cm beneath a lamp and left to
study. However, Chapman (1999) found that although warm up for 5 minutes. Pairs of temperature readings
translocation affected the distance moved by the snails, it were then measured: the temperature of the shell surface
did not result in any change in the direction at which the and the average temperature of the substrate surface at the
snails dispersed. perimeter of the snail. The procedure was repeated until all
75 animals were used. A one-way ANOVA was used to test
During the next four consecutive days (16 to 20 December for signifi cant differences among TD , TD and
Littoraria E. malaccana
2002), the distance between the locations of every marked TD . In order to maintain approximately equal
fi led E. malaccana
individual from the MHWS level was measured to the variances, two obvious outliers were removed prior to
nearest 0.1 cm. The mean tidal level occupied by each ANOVA analyses. The distance of the infrared thermometer
species on each of the four days was calculated. Crowe & from the surface was kept as constant as possible by
Underwood (1999) cited that upon disturbance, littorinids maintaining the laser spot at approximately the same size
are likely to require two days before resuming their natural during each measurement.
ways. Thus, four days would be suffi cient for the duration of
the translocation study. Data collected were analysed with
one-way Analysis of Variance (ANOVA) tests to ascertain Lethal temperatures study
whether the preferred zones occupied by the two species
differed signifi cantly. The Tukey’s multiple comparison test Littoraria, E. malaccana and E. vidua individuals with shell
was applied when ANOVA results were signifi cant. lengths between 7–11 mm were collected from the sloping
rock at Tanjung Chek Jawa in November 2002. Snails
were used within 24 hours of collection. The experiment
Effect of shell nodulation of E. malaccana involved 12 temperature treatments between 44 and 52°C
on heat refl ection (see below). For each temperature treatment, 30 littorinid
individuals (i.e., 10 Littoraria, 10 E. malaccana and 10 E.
Collection of data was carried out on two occasions: at vidua) were placed in an aluminium can containing 100ml
Tanjung Chek Jawa and in the laboratory. Temperature of artifi cial seawater. Heat treatment of the snails submerged
measured by a Raynger® ST™ infrared thermometer in seawater served to eliminate the effect of desiccation (see
(Raytek®) was taken as an indication of the amount of Evans, 1948). A plastic cover was placed over the can to
heat energy given off by the littorinid. The thermometer prevent water loss due to evaporation during heating. The
detects emitted energy (e.g., radiated heat energy from seawater was brought to the required temperature in a water
within the snail/gastropod) and refl ected heat energy (from bath from room temperature (22–23°C). The temperature
an external source e.g., the sun). At Tanjung Chek Jawa, the of the seawater was measured with “K” type thermocouple
average temperature of the shell of the rough periwinkle, probe connected to an AI 104 humidity temperature meter
E. malaccana, was measured using the average-function of (Azex Instruments (S) Pte. Ltd.). The probe was inserted
the infrared thermometer. The average temperature of the through a hole in the plastic cover. Once the required
rock surface, i.e., at the perimeter of the animal, was then temperature was attained, the time was noted and the
measured. Likewise, the temperatures of the shell surface seawater was maintained at a relatively constant temperature
of the smooth periwinkle, Littoraria, and the surrounding for an hour (the temperature was monitored and recorded
surface were measured under direct sunlight. A total of 55 at 15 minute intervals). This length of time was used by
pairs of temperature readings were recorded for individuals Fraenkel (1966) in a heat stress experiment conducted
of various sizes in each species. All individuals, whose shell on 12 species of intertidal prosobranchs in south-western
temperatures were measured, were selected such that they Japan. The time taken for the seawater to reach the required
were found on relatively smooth surfaces and not in any temperature ranged between 20 and 30 minutes. Monitoring
sort of pit or crevice. This was to ensure that measurements of seawater temperatures indicated that the seawater could
553
Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids
not be assumed to be at the temperature shown by the water calculated. Glutamate oxaloacetate transaminase (GOT)
bath as the temperature probe that was inserted into the activity (µmol NADH/min/g wet weight) was calculated
experimental container registered some fl uctuations. Hence, according to the following formula:
the temperature of each heat treatment was taken to be the
average temperature of the temperatures recorded during the (ΔA/min) (total volume) (volume of buffer
hour. The resultant 12 temperature treatments were 44.1°C, + wet weight of tissue) × 50
44.7°C, 45.6°C, 47.0°C, 47.4°C, 48.0°C, 48.4°C, 48.7°C, GOT =
49.5°C, 50.3°C, 50.8°C and 51.8°C. The temperature range 6.22 × sample volume × wet weight of tissue
selected was based on temperatures observed in the fi eld.
There were six replicates for each of the four temperature
After heat treatment, the littorinids were then transferred to treatments for all three littorinid taxa. GOT activity was
the centre of a holding tank containing artifi cial seawater converted to a percentage value based on the results
and an algae-covered rock. The tank was left in a normal of previous temperatures, as an indication of change in
external environment (i.e., out of the air-conditioned activity due to increase in temperature. Data were arcsine-
laboratory). At the end of 24 hours in the holding tank, transformed and analysed with one-way ANOVA. This
the number of live individuals was recorded. An individual was performed only on data for 50°C and 55°C, as most
was considered to be alive if it displayed foot attachment, of the data for 25°C and 45°C were greater than 100% and
had scaled up the sides of the tank, moved onto the rock or could not be arcsine-transformed. All statistical analyses
responded to gentle prodding of the foot. The percentage in this study were carried out using MINITAB (Release
mortalities obtained were converted into probit values and 12.21, 1998).
graphed against temperature. The 1-hour lethal temperature
(1h LT ) of each group was taken to be the temperature at
50
which there was 50% mortality. As it was not feasible to RESULTS
have a large sample size, statistical analyses of the 1h LT s
50
was not carried out. Distribution studies
At the vertical rock study site, Littoraria and E. malaccana
Heat stability of GOT were found in two distinct regions of the high intertidal zone
in June/July (Fig. 1a): Littoraria occupied the lower region
Six samples of tissues (each consisting approximately 0.5 g and E. malaccana, the higher region. The boundary between
fresh weight of pooled fl esh from a few individuals) of each the two regions was located approximately at 2.6 m (0.1 m
of the three littorinid taxa were frozen using liquid nitrogen below MHWS). The mean tide level (mean TL) occupied
and stored at -80°C. The frozen samples were pounded to by E. malaccana was 2.8 ± 0.2 m (range = 2.4 m to 3.6 m)
powder in liquid nitrogen, reweighed and homogenised while that of Littoraria was 2.0 ± 0.2 m (range = 1.8 m to
with thrice their weight of cold 0.05M Tris-HCl buffer 3.1 m) (Fig. 1a). In September 2002, the mean TL occupied
(from Sigma), pH 7.0. Homogenisation was done using a by E. malaccana increased to 3.0 m ± 0.3 m (range = 2.1
Polytron PT1300D homogeniser for a duration of 3 × 20 s m to 3.5 m), while that of Littoraria also increased to 2.3
with 20 s intervals. The homogenates were sonicated with ± 0.2 m (range = 1.9 m to 3.2 m) (Fig. 1b). The band of E.
a Heat Systems sonicator at 40% output power for one malaccana had a 0.6 m overlap with of the Littoraria band.
minute. The mixtures were centrifuged at 9000 rpm, 4 °C In December 2002, the overlapping region decreased to 0.3
for 10 min in a Beckman J2-MC refrigerated centrifuge. The m in width (Fig. 1c). E. malaccana was found between TL
supernatants were pipetted into clean test tubes, which were of 2.5 m to 3.6 m (mean TL = 3.2 ± 0.3 m) and Littoraria
kept in ice. Enzyme analyses were conducted within a few was found between TL of 2.0 m to 3.5 m (mean TL = 2.4
of hours of sample preparation using the GOT optimized ± 0.2 m) (Fig. 1c). At the sloping rock, E. malaccana also
aspartate-aminotransferase EC 2.6.1.1 UV-test kit (Sigma). occupied the highest region of the rock (range = 2.6m to
The sample used for enzyme analyses was a 50× dilution 2.8 m) while E. vidua individuals occupied a region that
of the extract (0.03 ml of the supernatant mixed with 1.47 overlapped with that of Littoraria (Fig. 2). The distribution
ml of Tris-HCl buffer, pH 7.0). Prior to enzyme analyses, of E. vidua extended slightly higher than the Littoraria zone
Reagent A (260 mmol/L aspartate, 104 mmol/L phosphate but was distinctly below that of E. malaccana.
buffer pH 7.4, 0.234 mmol/L NADH, 780 U/L MDH and
1560 U/L LDH) and the samples were incubated separately
at 25°C, 45°C, 50°C and 55°C for 30 minutes. After the Translocation experiment
30 min incubation, 0.1 ml of sample was added to 1.3 ml
of Reagent A and mixed by inversion. After a minute of The mean TL occupied by Littoraria on the four consecutive
incubation in a temperature controlled cuvette compartment days was slightly lower than the MHWS level of 2.7 m while
of a UV-1601 spectrophotometer with a CPS temperature E. malaccana and fi led E. malaccana were found at least
controller (Shimadzu), 0.125 ml of Reagent B (156 mmol/L 0.3 m above the MHWS level (Table 1). One-way ANOVA
2-oxoglutarate) was added and mixed. The absorbance of results showed that on all four days there was a signifi cant
the mixture was recorded eight times at 25 s intervals and difference (P < 0.05) among the mean TLs occupied by
the mean absorbance change per minute (ΔA/min) was the three species of littorinids (Table 2). Tukey’s multiple
554
THE RAFFLES BULLETIN OF ZOOLOGY 2009
Fig. 1. Kite diagrams showing the distributions of Littoraria spp. (LL) and Echinolittorina malaccana (EM) on a vertical rock at Tanjung Chek Jawa in
(a) June/July, (b) September, and (c) December 2002. (MHWS is represented by dashed line).
Fig. 2. Kite diagrams showing the distributions of Littoraria spp. (LL), Echinolittorina malaccana (EM) and E. vidua (EV) on a sloping rock (angle of
elevation of approximately 35.6°) at Tanjung Chek Jawa. (MHWS is represented by dashed line).
555
Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids
Table 1. Mean tide levels occupied by marked Littoraria spp. (Littoraria), Echinolittorina malaccana (E. malaccana) and fi led E. malaccana
(fi led E. malaccana) on four consecutive days (16 – 20 Dec.2002) at Tanjung Chek Jawa, Singapore.
Mean tide level ± S.D. (m)
Littoraria E. malaccana Filed E.
malaccana
Day 1 2.69 ± 0.20 3.11 ± 0.23 3.03 ± 0.23
Day 2 2.54 ± 0.24 3.11 ± 0.20 3.04 ± 0.23
Day 3 2.59 ± 0.25 3.14 ± 0.19 3.11 ± 0.26
Day 4 2.67 ± 0.21 3.16 ± 0.21 3.09 ± 0.21
Table 2. Results of one way ANOVA and Tukey’s multiple comparison for tide levels that were occupied by marked Littoraria spp.
(Littoraria), Echinolittorina malaccana (E. malaccana) and fi led E. malaccana (fi led E. malaccana) on four consecutive days (16 – 20
December 2002) at Tanjung Chek Jawa, Singapore. (*: signifi cant).
ANOVA
Tukey’s test
F df, df P
1 2
Day 1 29.99 2, 87 0.00 * Littoraria < fi led E. malaccana ≈ E. malaccana
Day 2 54.23 2, 87 0.00 * Littoraria < fi led E. malaccana ≈ E. malaccana
Day 3 52.41 2, 87 0.00 * Littoraria < fi led E. malaccana ≈ E. malaccana
Day 4 33.84 2, 87 0.00 * Littoraria < fi led E. malaccana ≈ E. malaccana
comparison tests showed that there was no significant 50.4°C (Fig. 3). Echinolittorina vidua had an intermediate
difference between the TLs occupied by fi led E. malaccana 1 h LT of approximately 48.1°C (Fig. 3).
50
and E. malaccana on the four days (P > 0.05) (Table 2).
Both E. malaccana and fi led E. malaccana were found at
a signifi cantly higher TL (P < 0.05) than Littoraria (i.e., Heat stability of GOT
Littoraria < fi led E. malaccana ≈ E. malaccana) (Table
2). Incubation of GOT at 45°C resulted in increased GOT
activity for all three species of littorinids with greatest
increase in GOT activity seen in E. malaccana (236.48 ±
Effect of shell nodulation of EM on heat refl ection 6.11%, Table 4). The maximum GOT activity for E. vidua
occurred after incubation at 50°C while those of Littoraria
Results from t-tests showed that TD and TD and E. malaccana were at 55°C (Fig. 4). ANOVA results
Littoraria E. malaccana
recorded at Tanjung Chek Jawa were both signifi cantly showed that after incubation at 50°C, the mean percentage
greater than 0°C (t = 3.36 and t = 7.61 respectively, P < GOT activity remaining for E. malaccana was signifi cantly
0.05 for both). The mean TD between rock surface and shell greater than that of E. vidua but not signifi cantly different
surface for E. malaccana and Littoraria were 0.55 ± 0.54°C from that of Littoraria (P < 0.05, E. vidua < Littoraria <
and 0.15 ± 0.34°C respectively. TD was signifi cantly E. malaccana). After incubation at 55°C, mean percentage
E. malaccana
greater than TD (W = 3942.5, P < 0.05). GOT activity remaining for E. malaccana was signifi cantly
Littoraria
greater than those from E. vidua and Littoraria, and that of
The ANOVA results of the laboratory heat experiment Littoraria signifi cantly greater than that of E. vidua (P <
showed that mean TD varied significantly with the 0.05, E. vidua < Littoraria < E. malaccana).
treatment group of littorinids in question (Table 3, P <
0.05). Tukey’s multiple comparison test showed that there
was no signifi cant difference between TD and TD DISCUSSION
Littoraria fi led
but TD was signifi cantly greater than both
E. malaccana E. malaccana
TD and TD (TD ≈ TD < During all three sampling periods, Littoraria (i.e., L.
Littoraria fi led E. malaccana Littoraria fi led E. malaccana
TD )(Table 3). articulata and L. strigata) was generally found below
E. malaccana
the MHWS level of 2.7 m. In consultation with the
Singapore Tide Tables and Port Information (Hydrographic
Lethal temperature study Department, 2001), it was estimated that the MHWS level
could be left dry for at least eight days during a two-week
Littoraria recorded the lowest 1h LT of approximately tidal cycle before the next high tide wetted the region.
50
47.5°C, and E. malaccana, the highest, at approximately Littoraria individuals may not be able to cope with the
physical stresses beyond the MHWS level during the
556
THE RAFFLES BULLETIN OF ZOOLOGY 2009
Table 3. Results of one-way ANOVA and Tukey’s multiple comparison of the variable, temperature difference (TD) between rock surface
and shell surface of Littoraria spp. (Littoraria), Echinolittorina malaccana (E. malaccana) and fi led E. malaccana in the laboratory.
(*: signifi cant).
ANOVA
V ariable Mean ± S.D. (°C)
Tukey’s test
F df1, df2 P
TD 0.128 ± 0.177
Littoraria
TD 0.300 ± 0.204 5.85 2, 72 0.004 * TD ≈ TD < TD
E. malaccana Littoraria fi led E. malaccana E. malaccana
TD 0.163 ± 0.174
fi led E. malaccana
aerial exposure. At the sloping rock, E. vidua was found Littorina granularis) and Littoraria (as L. undulata), Lam
to occupy a region distinctly below that of E. malaccana (1980) found that E. malaccana was most tolerant to
and it overlapped with the upper region in which Littoraria desiccation at 38 ± 1ºC, a trait that partly accounts for its
were found. successful survival above the MHWS level.
Echinolittorina malaccana was generally found above The distributions of Littoraria and E. malaccana were
and at the MHWS level. Lam (1980) also reported that E. generally distinct from each other, with the former found
malaccana (as Tectarius malaccensis) inhabited the splash at a lower level than the latter. This zonation observed
zone above MHWS on a man-made breakwater. A similar at Tanjung Chek Jawa was verifi ed by the translocation
distribution pattern was observed by Ohgaki (1988b) study, whereby displaced individuals of Littoraria and E.
for another rough periwinkle, Nodilittorina exigua, in malaccana returned to their preferred regions on the shore.
Shirahama, Japan. Ohgaki (1988b) suggested that N. exigua Such movements were also observed in Austrolittorina
remained above and around the MHWS level to escape unifasciata (as L. unifasciata) and Nodilittorina pyramidalis
harsh conditions (e.g., strong wave action) that exist lower in New South Wales, Australia by Chapman (1999). In
down the shore. In addition, these nodulated littorinids addition, physical removal of shell nodules in E. malaccana
are adapted for survival under prolonged aerial exposure. did not result in a change of preference for a tide level, i.e.,
Among E. malaccana (as T. malaccensis), E. vidua (as the fi led E. malaccana did not move to a level signifi cantly
lower or higher than that of their unfiled counterparts.
However, results from the laboratory experiment showed
that fi led Echinolittorina shells and smooth Littoraria shells
were both warmer than nodulated shells, as evidenced by
the mean TD being signifi cantly greater than both
E. malaccana
TD and TD . This suggests that although
fi led E. malaccana Littoraria
shell ornamentation helps to reduce shell temperature, it is
probably not a major factor in determining the choice of
tidal level at which the species remained.
Rough, nodulated shells reflect more heat energy
(Nybakken, 2001). The higher TD of the rough periwinkle,
E. malaccana, observed in this study may be due to this
reason, as the infrared thermometer senses refl ected heat
energy (refer to Raynger® STTM manual). The signifi cant
difference between TD and TD at Tanjung
Littoraria E. malaccana
Chek Jawa suggested that the nodulation on E. malaccana
helped in reducing the amount of heat transmitted into the
body of the littorinid. This is because the more heat energy
is refl ected, less is absorbed and transmitted (Jones, 2000).
Another factor that may account for the difference in TDs
is colour, which affects absorption and refl ection of heat.
According to Vermeij (1973), a light-coloured surface
absorbs less radiation from visible light. Many high shore
gastropods (e.g., Nerita peleronta, Tectarius muricatus and
Littorina unifasciata antipodium) are light coloured while
gastropods at lower shores have darker shells (Nyabakken,
2001; Vermeij, 1973). The nodules on E. malaccana are
of a much lighter colour (pale yellow) than the rest of the
Fig. 3. Probit response curves of Littoraria sp. (●), Echinolittorina vidua shell (dark brown). This colouration may further enhance
(●) and E. malaccana (■), from which the 1h LT values are obtained.
50
557
Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids
Table 4. Mean percentage glutamate oxaloacetate transaminase (GOT) activity remaining for Littoraria sp. (Littoraria), Echinolittorina
vidua and E. malaccana after incubation at 45°C, 50°C and 55°C.
Mean percentage GOT activity ± S.D. (%)
Species

25°C 45°C 50°C 55°C
Littoraria 100.00 ± 0.00 176.32 ± 69.78 112.27 ± 56.57 67.87 ± 7.95
E. vidua 100.00 ± 0.00 191.99 ± 10.39 98.41 ± 9.39 47.27 ± 8.73
E. malaccana 100.00 ± 0.00 236.48 ± 6.11 101.94 ± 22.97 97.59 ± 33.69
heat refl ection by the nodules. Filed shells were much darker an almost terrestrial life above and about MHWS such that
in colour than intact conspecifi cs. McQuaid and Schermann it is unable to cope with prolonged immersion. Interspecifi c
(1988) reported that the pale-shelled L. a. africana had competition between Littoraria and E. malaccana could
a lower body temperature than the dark brown morph L. also infl uence the zonation pattern. However, more studies
a. knysnaensis. They also found that there was a higher would have to be conducted to elucidate the interaction
mortality rate among individuals of the two sub-species that between the two genera at the study site.
were painted black.
The 1h LT s of the three taxa of littorinids in this study
50
The preference for a higher or lower shore level may increased with their relative position on the shore. Littoraria
likely be due to physiological adaptations. McMahon found lowest on the shore had the lowest 1h LT of
50
(2001) hypothesized that mid-shore gastropods had 47.5°C while the inhabitant of the highest level on the
modified respiratory systems for aerial and aquatic gas shore, E. malaccana, had the highest 1h LT of 50.4°C.
50
exchange, which disabled them from surviving in aquatic The 1h LT of E. vidua (48.1°C) reflected its position
50
environments. Likewise, E. malaccana may have adapted to on the shore relative to E. malaccana and Littoraria, i.e.,
between the two genera. The increase in thermal tolerance
with increase in position on the shore is consistent with
fi ndings of other researchers. Southward (1958) found that
the high-shore top-shells Monodonta lineata and Gibbula
umbilicalis tolerated higher temperatures that G. cineraria
and Callipstoma zizyphinum, which inhabited low water.
Fraenkel (1966) observed that N. pyramidalis at the upper
part of the intertidal zone was most heat resistant, while
Chlorostoma argyrostoma lischkei collected from the
water’s edge was least resistant. In addition, Wolcott (1973)
reported that two littoral fringe limpets (Acmaea digitalis
and A. scabra) maintained higher tissue temperatures that
two other species (A. pelta and A. testudinalis scutum)
present in the eulittoral zone.
The 1h LT value for N. pyramidalis in Japan was
50
determined by Fraenkel (1966) to be 48.5°C, which was
lower than the 1h LT of 50.4°C obtained in this study.
50
His samples were heated in air instead of seawater, which
may have resulted in some degree of desiccation (see Evans,
1948) thus affecting the thermal tolerance of the littorinid.
Wolcott (1973) reported that removal of body water by
desiccation reduced the thermal tolerance of the limpet A.
digitalis by about 3°C.
In addition, the different 1h LT may be due to the
50
geographical location in which the species inhabits.
Thermal tolerance is reportedly negatively correlated to
latitude (Fraenkel, 1968; McMahon, 2001). This may be a
result of the higher level of solar radiation all year round
in the tropics when compared with that of temperate areas
(Moore, 1972). Southward (1958) reported that Littorina
Fig. 4. Mean glutamate oxaloacetate transaminase (GOT) activities (µmol
NADH/min/g wet weight) of Littoraria sp., Echinolittorina malaccana and neritoides found in North Africa was more heat tolerant than
E. vidua from Tanjung Chek Jawa at four temperatures: 25 °C, 45 °C, L. saxatilis found all around Britain. Of the 60 gastropods
50 °C and 55 °C. (Vertical bars represent standard errors). Littoraria sp.
collected from various localities (e.g., Australia, England,
●––––●; Echinolittorina malaccana, ●........●; E. vidua ■-------■.
558
THE RAFFLES BULLETIN OF ZOOLOGY 2009
Hong Kong, Jamaica, Texas), McMahon (2001) found that Chapman, M. G., 1999. Assessment of variability in responses
Nodilittorina ziczac from Jamaica (18°N) had the highest of intertidal periwinkles to experimental transplantations.
mean heat coma temperature (HCT) of 46.9°C. Littorina Journal of Experimental Marine Biology and Ecology, 236:
171–190.
mariae found in Robin Hoods Bay, England (54°N) had
the lowest mean HCT of 26.4°C. Similarly, N. pyramidalis Cleland, J. D. & R. F. McMahon, 1989. Upper thermal limit of nine
at Wakayama, Japan (34°N) had a slightly lower thermal intertidal gastropod species from a Hong Kong rocky shore in
relation to vertical distribution and desiccation associated with
tolerance than E. malaccana found Tanjung Chek Jawa,
evaporative cooling. Proceedings of the Second International
Singapore (approx. 1°N). Tropical E. malaccana possibly
Marine Biological Workshop: The Marine Flora and Fauna
adapted to higher temperatures near the equator. This
of Hong Kong and Southern China, Hong Kong. Hong Kong
species is found on rocks that may be heated to temperatures
University Press, Hong Kong, pp. 1141–1152.
of 45°C to 50°C (Lim & Fahmy, 2008). McMahon (2001)
Cooper, A. J. L. & A. Meister, 1985. Metabolic significance
reported a HCT of 46.3°C for E. malaccana in Hong Kong
of transamination. In: Christen, P. & D. E. Metzler (eds.),
(22°N) (c.f. 1h LT 50.4°C recorded in this study). This
50 Transaminases. Wiley, New York. Pp. 533–563.
is further evidence for the resilience of this species under
Crowe, T. P. & A. J. Underwood, 1999. Differences in dispersal
high temperatures.
of an intertidal gastropod in two habitats: the need for and
design of repeated experimental transplantation. Journal of
Enzyme stability, such as that observed for GOT in E.
Experimental Marine Biology and Ecology, 237: 31–60.
malaccana, may account for the relatively high heat
Evans, R. G., 1948. The lethal temperatures of some common
tolerance of this littorinid in the current study. Glutamate
British littoral molluscs. Journal of Animal Ecology, 17:
oxaloacetate transaminase is required for the regulation of
165–173.
carbon and nitrogen fl ow in various biochemical pathways
Fraenkel, G., 1966. The heat resistance of intertidal snails at
(Cooper & Meister, 1985). An enzyme’s function is highly
Shirahama, Wakayama-ken, Japan. Publications of the Seto
dependent on its structure. The stability of this structure is
Marine Biological Laboratory, 14: 185–195.
temperature-sensitive (Somero, 1978), denaturing at high
Fraenkel, G., 1968. The heat resistance of intertidal snails at
temperatures. Among Littoraria sp., E. malaccana and
Bimini, Bahamas; Ocean Springs, Mississipi; and Woods
E. vidua, the percentage of GOT activity remaining after
Hole, Massachusetts. Physiological Zoology, 41: 1–13.
incubation at 55 °C was highest for E. malaccana. This
Gould, A. A., 1859. Descriptions of new species of shells brought
indicated that GOT from E. malaccana was most stable,
home by the North Pacifi c Exploring Expedition. Proceedings
even at a temperature above its 1h LT (50.4°C). Somero
50 of the Boston Society of Natural History, 7: 138–142.
(1978) reported that enzymes denature at temperatures that
Hydrographic Department, 2001. Singapore Tide Tables and Port
are usually higher than the upper lethal temperature of the
Information. 2002. Hydrographic Department, Maritime and
species. After incubation at this temperature, the activity
Port Authority of Singapore. 239 pp.
of GOT from Littoraria and E. vidua fell sharply, a likely
Hull, S. L., J. Grahame & P. J. Mill, 1999. Heat stability
result of denaturation of the enzymes. Hull et al. (1999)
and activity levels of aspartate aminotransferase and
also found that enzymes of high shore animals (e.g., high
alanine aminotransferase in British Littorinidae. Journal of
shore L. saxatilis and L. arcana) were more stable, i.e., had Experimental Marine Biology and Ecology, 237: 255–270.
higher activity rates at 56°C, than those of low shore groups
Jones, H.R.N. 2000. Radiation heat transfer. Oxford University
(e.g., L. obtusata, L. neglecta and L. littorea). This trend of
Press, New York. 83 pp.
increased stability with increased height on the shore was
Lam, Y. K., 1980. Some studies on the East Coast breakwater
also evident within a single species, L. saxatilis, found at
fauna, with particular reference to the vertical distribution
high and mid-shore levels.
of the periwinkles (Littorinidae) and their tolerances to
oil pollution. Unpublished Honours thesis, University of
Singapore, Singapore. Pp 97 + 9 appendices.
ACKNOWLEDGEMENTS
Lim, S. S. L., 2008. Body posturing in Nodilittorina pyramidalis
and Austrolittorina unifasciata (Mollusca: Gastropoda:
The authors thank the Nature Conservation Branch Littorinidae): a behavioural response to reduce heat stress.
(National Parks Board) for the permit to conduct research In: Davie, P. J. F. & J. A. Phillips (eds.), Proceedings of
and to collect specimens at Pulau Ubin Nature Park. The the Thirteenth International Marine Biological Workshop,
staff of the National Parks Board Offi ce at Pulau Ubin is The Marine Fauna and Flora of Moreton Bay, Queensland.
Memoirs of the Queensland Museum – Nature, 54: 339–347.
acknowledged for their assistance in fi eld logistics. Financial
support for this study was provided by a grant (RP 19/97 Lim, S. S. L. & M. Fahmy, 2008. Is ‘standing’ posture an effective
LSL) from the National Institute of Education, Nanyang behavioural adaptation to reduce heat stress in Echinolittorina
malaccana? Abstracts of the IX International Symposium on
Technological University to the second author (SSLL).
Littorinid Biology and Evolution, University of Vigo, Spain,
p. 17.
LITERATURE CITED McMahon, R. F., 1990. Thermal tolerance, evaporative water
loss, air-water oxygen consumption and zonation of intertidal
prosobranchs: a new synthesis. Hydrobiologia, 193: 241–
Bingham, F. O., 1972. The mucus holdfast of Littorina irrorata
260.
and its relationship to relative humidity and salinity. The
Veliger, 15: 48–50.
559
Lee & Lim: Vertical zonation and heat tolerance of Singapore littorinids
McMahon, R. F., 2001. Acute thermal tolerance in intertidal Philippi, R. A., 1847. Abbildungen und Beschreibungen neuer
gastropods relative to latitude, superfamily, zonation and oder wenig gekannter Conchylien. Vols 2, 3. Theodor Fischel,
habitat with special emphasis on the Littorinoidea. Journal of Cassel; Vol. 3: 1–50.
Shellfi sh Research, 20: 459–467.
Raffaelli, D. & S. Hawkins, 1996. Intertidal Ecology. Chapman &
McMahon, R. F. & W. D. Russell-Hunter, 1977. Temperature Hall, New York. 356 pp.
relations of aerial and aquatic respiration in six littoral snails
Reid, D. G., 1986. The littorinid molluscs of mangrove forests in
in relation to their vertical zonation. Biological Bulletin, 152:
the Indo-Pacifi c region: the genus Littoraria. British Museum
182–198.
(National History), London. Pp. 227 + xv.
McQuaid, C. D., & P.A. Schermann, 1988. Thermal stress in a high
Reid, D. G., 2007. The genus Echinolittorina Habe, 1956
shore intertidal environment: morphological and behavioural
(Gastropoda: Littorinidae) in the Indo-West Pacifi c Ocean.
adaptations of the gastropod Littorina africana. In: Chelazzi,
Zootaxa, 1420: 1–161.
G. & M. Vannini (eds.), Behavioural Adaptation to Intertidal
Life. Plenum Press, New York. Pp. 213–224. Somero, G. N., 1978. Temperature adaptation of enzymes: biological
optimization through structure-function compromises. Annual
MINITAB, INC., 1998. MINITAB User’s Guide 2: Data analysis
Review of Ecology and Systematics, 9: 1–29.
and quality tools. Release 12 for Windows. State College, PA,
USA. 22 Chapters [paginated by chapters]. Southward, A. J., 1958. Note on the temperature tolerances
on some intertidal animals in relation to environmental
Moore, H. B., 1972. Aspects of stress in the tropical marine
temperatures and geographical distribution. Journal of the
environment. Advances in Marine Biology, 10: 217–269.
Marine Biological Association of the United Kingdom, 37:
Newell, R. C., 1979. Biology of intertidal animals. Marine 49–66.
Ecological Surveys Ltd., U.K. Pp. 781 + ix.
Tan, K. S., 1988. The distribution of littorinids and movement
Nybakken, J.W., 2001. Marine biology: an ecological approach. tracking of Nerita lineata in an intertidal drain. Unpublished
Benjamin Cummings, San Francisco. 516 pp. Honours thesis, National University of Singapore, Singapore.
Pp 68 + iii.
Ohgaki, S., 1988a. Rain and the distribution of Nodilittorina exigua
(Dunker) (Gastropoda: Littorinidae). Journal of Experimental Vermeij, G. J., 1973. Morphological patterns in high-intertidal
Marine Biology and Ecology, 122: 213–223. gastropods: adaptive strategies and their limitations. Marine
Biology, 20: 319–346.
Ohgaki, S., 1988b. Vertical migration and spawning in Nodilittorina
exigua (Gastropoda: Littorinidae). Journal of Ethology, 6: Wolcott, T. G., 1973. Physiological ecology and intertidal zonation
33–38. in limpets (Acmaea): a critical look at “limiting factors”.
Biological Bulletin, 145: 389–422.
Petraitis, P. S., 1982. Occurrence of random and directional
movements in the periwinkle, Littorina littorea (L.). Journal of
Experimental Marine Biology and Ecology, 59: 207–217.
Philippi, R. A., 1846. Descriptions of a new species of Trochus,
and of eighteen new species of Littorina, in the collection of
H. Cuming, Esq. Proceedings of the Zoological Society of
London, 1845: 138–143.
560